Comparison of chemical compositions, antioxidant activities, and acetylcholinesterase inhibitory activities between coffee flowers and leaves as potential novel foods

Abstract This study aimed to compare chemical compositions, antioxidant activities, and acetylcholinesterase inhibitory activities of coffee flowers (ACF) and coffee leaves (ACL) with green coffee beans (ACGB) of Coffea Arabica L. The chemical compositions were determined by employing high‐performance liquid chromatography–mass spectroscopy (HPLC–MS) and gas chromatography–mass spectroscopy (GC–MS) techniques. Antioxidant effects of the components were evaluated using DPPH and ABTS radical scavenging assays, and the ferric reducing antioxidant power (FRAP) assay. Their acetylcholinesterase inhibitory activities were also evaluated. The coffee sample extracts contained a total of 214 components identified by HPLC‐MS and belonged to 12 classes (such as nucleotides and amino acids and their derivatives, tannins, flavonoids, alkaloids, benzene, phenylpropanoids, and lipids.), where phenylpropanoids were the dominant component (>30%). The contents of flavonoids, alkaloids, saccharides, and carboxylic acid and its derivatives in ACF and ACL varied significantly (p < .05) compared to similar components in ACGB. Meanwhile, 30 differentially changed chemical compositions (variable importance in projection [VIP] > 1, p < .01 and fold change [FC] > 4, or <0.25), that determine the difference in characteristics, were confirmed in the three coffee samples. Furthermore, among 25 volatile chemical components identified by GC–MS, caffeine, n‐hexadecanoic acid, 2,2′‐methylenebis[6‐(1,1‐dimethylethyl)‐4‐methyl‐phenol], and quinic acid were common in these samples with caffeine being the highest in percentage. In addition, ACL showed the significantly highest (p < .05) DPPH radical scavenging capacity with IC50 value of 0.491 ± 0.148 mg/ml, and acetylcholinesterase inhibitory activity with inhibition ratio 25.18 ± 2.96%, whereas ACF showed the significantly highest (p < .05) ABTS radical scavenging activity with 36.413 ± 1.523 mmol trolox/g Ex. The results suggested that ACL and ACF had potential values as novel foods in the future.


| INTRODUC TI ON
Coffea is a genus of the family Rubiaceae, used to make coffee beverages, which are on the top among the three beverages in the world, due to their rich and complex flavor and medicinal values. As an important plantation crop, Coffea is grown in more than 80 countries around the world (Godos et al., 2014). Coffea cultivating areas cover about 10.6 million ha of land and are mainly distributed in the tropics, such as Brazil, Colombia, Venezuela, Paraguay, Indonesia, India, Ethiopia, and Mexico (Silva et al., 2013). A large number of coffee by-products including grounds, silver skins, husks, flowers, and leaves of coffee are produced by the global coffee plantation and processing industries. Some coffee by-products are already used as food in Europe and non-EU-member countries, or have been applied for authorization as novel food already (Klingel et al., 2020).
Coffee by-products are beneficial for human health, owing to the presence of some natural bioactive compounds, such as phenolic acids, flavonoids, terpenes, and alkaloids. These bioactive compounds show antioxidant and hepato-and neuro-protective activities Martinez-Saez & Dolores del Castillo, 2019;Rebollo-Hernanza et al., 2019). Coffee by-products, which can be important sources of natural functional compounds in the future, will contribute to the development of functional compounds and circular economy (Comunian et al., 2021;Panwar et al., 2021). In addition, coffee by-products can be recycled to produce value-added products in bioenergy segment. However, the increasing production of solid residues of coffee by-products originating from annual coffee production has brought about environmental concerns.
Coffee flowers, as the primary coffee by-products are usually abandoned in coffee cultivation, have received growing public attention and research interests for their potential human health benefits due to their various phytochemicals (Nguyen et al., 2019;Pinheiro et al., 2021). Pinheiro et al. (2021) suggested that the flowers of C. Arabica and C. Conilon possessed antioxidant properties. Thus, coffee flowers can be potentially used for research and development of bioactive compounds focusing on human health. Coffee leaves were widely used as medicine and beverages in some countries and regions consuming tea as their primary beverage .
The health benefits of bioactive components in coffee leaves have been reported by several researchers (Campa et al., 2012;Chen et al., 2018). The chemical composition of coffee leaves consists of alkaloids, flavonoids, phenolic acids, terpenes, and so on, responsible for antioxidation, anti-inflammatory, antitumor, antidiabetic, and neuroprotective activities .
Studies had found that oxidative stress is one of the most important mechanisms of cellular senescence and increased frailty, resulting in several age-linked, noncommunicable diseases (Martemucci et al., 2022). Antioxidants can protect cells against free radical damage, as well as help in reducing the risk of many chronic diseases, such as Alzheimer's disease (AD) (Singh et al., 2020). In addition, AD is related to the decrease of the neurotransmitter acetylcholine (ACh) levels (Zavala-Ocampo et al., 2022). Based on the cholinergic hypothesis cholinesterase inhibitors are used to re-establish the levels of acetylcholine in the brain (Sahibzada et al., 2022). Therefore, antioxidant and acetylcholinesterase inhibitory activities are the basis for further studies in the development of therapies for neurodegenerative disorders. Eicosanoyl-5-hydroxytryptamide, caffeic acid, and caffeine both showed a beneficial therapeutic effect in a rat model of sporadic AD. (Asam et al., 2017;Rezg et al., 2008;Zeitlin et al., 2011).
Yunnan province, the main coffee plantation area in China, undertakes 99% of Chinese coffee plantations. A large number of coffee flowers and leaves were discarded annually. The aim of this study is to provide data for further development and utilization of coffee by-products as well as to enhance the value of coffee. The experimental design of the study is shown in Figure 1. The identification of chemical compounds in coffee flowers (ACF), leaves (ACL), and green coffee beans (ACGB) from C. Arabica was done by high-performance liquid chromatography-mass spectroscopy (HPLC-MS) and gas chromatography-mass spectroscopy (GC-MS).

| Plant material and reagents
Coffee flowers, coffee leaves, and fresh coffee cherries from C. Arabica were harvested and collected from Baoshan City, Yunnan (p < .05) DPPH radical scavenging capacity with IC 50 value of 0.491 ± 0.148 mg/ml, and acetylcholinesterase inhibitory activity with inhibition ratio 25.18 ± 2.96%, whereas ACF showed the significantly highest (p < .05) ABTS radical scavenging activity with 36.413 ± 1.523 mmol trolox/g Ex. The results suggested that ACL and ACF had potential values as novel foods in the future.

K E Y W O R D S
acetylcholinesterase inhibitory activity, antioxidant activity, chemical composition, coffee flower, coffee leaf Province, China. Coffee flowers and leaves were crushed and dried at room temperature. Green coffee beans were obtained by wet processing. The voucher specimens (No. ACBS1-3) were obtained from College of Science, Yunnan Agricultural University.
High-performance liquid chromatography grade methanol and acetonitrile were sourced from Merck KgaA, and formic acid was purchased from Xiya Reagent.

| Sample preparation
The dried samples were extracted using an ultrasound-assisted extraction method as follows: 25.0 g of the powdered samples were extracted with 200 ml of 80% MeOH aqueous (V/V) for 30 min. After extraction, the resulting solution was filtered with a filter paper. The residue obtained was washed with 100 ml of MeOH subsequently and was extracted again. The filtrates were then combined and concentrated using a YaRong rotary evaporator (Shanghai, China) at 50°C and finally freeze-dried. Afterward, the extracts were redissolved and stored at 4°C.

| HPLC-MS analysis
Sample extracts were analyzed with Q-Exactive HF mass spectrometer and Thermo Ultimate 3000LC. Chromatographic separation of the sample components was achieved a Zorbax Eclipse C18 column (1.8 μm × 0.25 mm × 100 mm). The mobile phases used were a mixture of water with 0.1% (V/V) formic acid (A) and acetonitrile (B). The gradient flow started with 5-30% B in 2 min, then 30% B at 7 min; followed by a linear increase to 78% B in 5 min, then 78% B at 14 min; followed by a linear increase to 95% B in 3 min, then 95% B at 17 min.
The gradient returned to 5% B at 21 min, leading to a 25 min total run time. The flow rate was 0.3 ml min −1 with a typical injection volume of 2 μl. The column temperature was maintained at 30°C. Mass spectrometry was performed with ESI ionization: full scan MS-DDMS2 F I G U R E 1 Experimental design acquisition mode; an ion transfer tube temperature of 350°C; spray voltage of 3.5 kV (+)/3.5 kV (−); sheath gas 45 arb; auxiliary gas 15 arb; a 100-1500 mass/charge m/z ratio range; resolution of 120,000 (full scan) and 60,000 (DDMS2).

| GC-MS analysis
Sample extracts were analyzed with a Thermo Scientific TSQ-8000 triple quadrupole mass spectrometer and a Trace GC 1300 gas chromatograph, which was equipped with a TriPlus AI 1310 autosampler (Thermo Fisher Scientific, San Jose, CA). Chromatographic

| DPPH scavenging capacity of the coffee samples
The DPPH scavenging capacity of the coffee samples was determined according to the literature method of DPPH assay (Deng et al., 2011;Hu et al., 2019): Rutin was used as a positive control. Sample mixtures were prepared by mixing 3.9 ml of DPPH (0.075 mM) and 0.1 ml of samples of different concentrations (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 0.1 mg/ml) and evaluated at 517 nm. The inhibition (I) was calculated using Equation (1) where A s is the mixture of samples and DPPH, A o is the DPPH. Trolox was used as a standard (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 [×10 −6 mmol]) and the ABTS radical scavenging capacity was calculated by a calibration curve given by Equation (2)

| FRAP assay of coffee samples
The FRAP assay was conducted using the reference method of Amamcharla and Metzger (2014). A mixture of 3.0 ml FRAP working reagent, prepared using TPTZ and FeCl 3 , was mixed with 100 μl sample solution and 300 μl deionized water. The sample mixture thus prepared was evaluated at 595 nm after being incubated at 37°C for 30 min. The Tacrine was used as a positive control with a final concentration of 0.333 μM. All the reactions were performed in triplicate. The percentage inhibition (I) was calculated using Equation (4) where A 0 is the activity of the enzyme without test extracts and A s is the activity of enzyme with test extracts.

| Statistical analysis
To establish the orthogonal partial least squares discriminant analysis (OPLS-DA) model, a permutation analysis was carried out on the data with the number of tests set to 200; the differences between the two groups of data were analyzed as a whole to obtain the volcanic maps and variable importance in projection (VIP) prediction value distributions. The chemical compositions with VIP > 1, p < .01 and fold change (FC) > 4, or <0.25 were designated as significantly changed compounds. One-way analysis of variance (ANOVA) with the least-significant difference (LSD) method (p < .05) was applied to compare inhibition shown by different samples. (1)

| Chemical compositions of the coffee samples
The results of HPLC-MS were processed and analyzed by Compound  of chemical compositions. Phenylpropanoids were identified to be the highest percentages out of 12 classes of chemical components in three samples, which ranged from 37.977% to 61.683%. Moreover, the percentages of phenylpropanoids in ACF and ACL were lower than in ACGB. Flavonoids were identified as primary components in ACL and ACF, and the percentages were 20.51% and 6.82%, respectively; while it was only 0.021% in ACGB. Meanwhile, the percentage of alkaloids in ACL was 12.96%, which was higher than ACGB (9.24%). However, the value of alkaloids in ACF was 6.20% which was lower than ACGB. Compared with ACF and ACL, the percentages  (4, 5, 15, 17, 20, 21, and 22) were unclear for their low contents.
Peak 9 was found to be the primary component in these three coffee samples with high intensities and was confirmed to be as caffeine accounting for the maximum area in all compounds. Compared to ACGB, ACL was richer in volatile compounds as 18 volatile compounds were detected in ACL; then there were 12 in ACGB, and 9 in ACF, respectively. These compounds are marked in Figure 6d. Four compounds were common in all the three coffee samples namely

| Antioxidant activity
The three models of testing the antioxidant activities of three coffee samples were based on different principles and the results are shown in

| Acetylcholinesterase inhibitory activity
Compared with Tacrine, ACL showed weak acetylcholinesterase inhibitory activity with an inhibition ratio of 25.18 ± 2.96%.

| DISCUSS ION
The chemical constituents of coffee, which are the basis of different biological activities of coffee and contribute to the characteristic flavor, are large in number, including alkaloids, phenolic acids, flavonoids, etc. (Shen et al., 2020). Substantiating the information available in literature, caffeine and chlorogenic acid were confirmed in this study as the main classic compounds in coffee.
Thirty distinct characteristic compounds obtained from these three coffee samples would help in the rapid analysis of the coffee samples.
Terpenes as a type of characteristic constituents in C. Arabica include the skeletons of ent-kaurane, kahweol, villanovane diterpenoid, entkaurane diterpenoid glucosides, dammarane, and pentacyclic triterpene (Shen et al., 2020). However, kahweol belonging to heterocyclic compounds was confirmed as the only terpene in this study. This may be attributed to the fact that new compounds with unknown structures are usually recovered in low concentrations; hence, they are not included in Thermo mzCloud and Thermo mzValut data.
The antioxidant properties of food products are considered as parameters of nutritional quality (Carlsen et al., 2010;Yang et al., 2011). Coffee contains multiple active components, including caffeine, phenolic acids, and flavonoids. The introduction of coffee by-products as a novel food in the food sector still needs many efforts. Pinheiro et al. (2021)  In addition, coffee leaves have been used to prepare tea-like drinks through leaf steaming, rolling, and drying production methods for a long time (Ratanamarno & Surbkar, 2017). Alternatively, the coffee leaves are also fermented and roasted as well as used as medicine in some originating countries. Because they contain multiple bioactive compounds including terpenes, tannins, phenolic acids, flavonoids, phytosterols, and carotenoids, which are related to their diverse potential bioactive effects. Moreover, coffee leaves were determined as a traditional food by the EFSA (European Food Safety Authority) in the context of a novel food notification from a third country. These results provide sufficient support to the use of ACL and ACF as novel potential food materials.
Moreover, many chemical compounds, such as flavonoids, polysaccharides, triterpenoids, also have acetylcholinesterase inhibitory activity (Li et al., 2020;Liu et al., 2019;Xu et al., 2022). In the same extraction conditions, ACL was richer in chemical compounds compared to ACGB and ACF. Therefore, ACL showed higher activity than ACGB and ACF. However, the potentially active compounds were not pure and hence further studies are needed.

CO N FLI C T O F I NTE R E S T
All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request from the authors: The data that support the findings of this study are available from the corresponding author upon reasonable request.